Calcific aortic valve disease (CAVD) is a major cause of aortic stenosis (AS). By age 65, ~2% of individuals develop CAVD, which, if untreated, leads to life threatening left ventricular dysfunction and heart failure. Patients with type II diabetic mellitus have a heightened incidence and risk for CAVD compared to those without. Currently there are no pharmacological therapies to improve the outcomes of CAVD. Aortic valve replacement remains the main treatment, though at high cost and significant risk in certain patients. The lack of drug therapies reflects major weaknesses in scientific knowledge of the definitive causes and mechanisms of AS in CAVD, partly due to lack of appropriate animal and cell culture models. Our scientific premise is that progressive calcification is a key cell-mediated process leading to AS in CAVD, and that identification of molecules and mechanisms regulating this process will aid in developing pharmacotherapies and targeting strategies to prevent AS, while minimizing skeletal toxicity. Our preliminary data show for the first time that inhibition of osteochondrogenic differentiation via deletion of the procalcific transcription factor, Runx2, in valve interstitial cells (VICs) improves valve function in a new diabetic model of CAVD. Furthermore, previous studies as well as our own preliminary data, suggest that Sox 9 promotes reparative processes that mediate valve sclerosis and oppose procalcific processes, and that the inflammatory mediator, TNF?, controls the balance between reparative/sclerotic vs. calcific/stenotic processes in CAVD. The overall hypothesis of the proposal is that Sox9 mediates ECM deposition and valve repair early following valve injury, which contributes to valve sclerosis. As disease progresses, inflammatory TNF? promotes Runx2 expression, phosphorylation, nuclear translocation and binding to Sox9, counteracting Sox9 transactivation, and thereby directing procalcific osteoblastic and/or hypertrophic chondrocytic phenotype, which drives calcification and AS. The aims of the proposal address key unanswered questions: What are the lineages of VICs in CAVD that contribute to repair of valve injury versus formation of mineralized cartilage and bone How do these lineages contribute to sclerosis versus stenosis? Can these lineages be targeted to prevent calcification and AS and avoid bone toxicity? Do key osteochondrogenic transcription factors, such as Sox9 and Runx2, control the switch from fibrotic repair (sclerosis) to calcific stenosis, respectively, during CAVD? Do inflammatory and/or disease- specific cytokines, such as TNF?, modulate these factors in VIC, thereby promoting differentiation towards a procalcific path and stenosis? Can preventing or turning off the procalcific osteochondrogenic switch improve valve function, offering us potential therapeutic targets? Answers to these questions could help with earlier detection, as well as improved biomarkers and targeted anticalcific therapies for CAVD, which could ultimately lead to improved treatments for CAVD.